Retroviral and lentiviral vectors

ABSTRACT

The present invention provides a retroviral or lentiviral vector having a viral envelope which comprises: (i) a mitogenic T-cell activating transmembrane protein which comprises a mitogenic domain and a transmembrane domain; and/or (ii) a cytokine-based T-cell activating transmembrane protein which comprises a cytokine domain and a transmembrane domain, wherein the mitogenic or cytokine-based T-cell activating transmembrane protein is not part of a viral envelope glycoprotein. When cells such as T-cells of Natural Killer cells are transduced by such a viral vector, they are simultaneously activated by the mitogenic T-cell activating transmembrane protein and/or the cytokine-based T-cell activating transmembrane protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/554,499, filed Aug. 30, 2017, which is a U.S. National Phase ofInternational Application No. PCT/GB2016/50537, filed Mar. 1, 2016,which claims priority to Great Britain Application No. 1503500.9, filedMar. 2, 2015.

The present invention relates to retroviral and lentiviral vectors andcells for their production. The vectors may be used for transducingcells, such as T-cells. In particular, the invention relates retroviralor lentiviral vectors capable of both transducing and activating a cell,such as a T cell.

BACKGROUND TO THE INVENTION

The generation of engineered T-cell products typically requiresstimulation with a mitogen followed by transduction with an integratingvector, such as a lentiviral vector or a retroviral vector.

A widely used approach is to add soluble mitogenic monoclonal antibodies(mAb), such as anti-TCR/CD3 and anti-CD28, to the cell culture. Analternative approach is to attach anti-TCR/CD3 mAb along with anti-CD28mAb to a bead. The surface of the bead has improved T cell activatingproperties compared to the soluble antibodies alone.

In addition cytokines (e.g. IL2, IL15 or IL7) are commonly added to thecell culture.

These mitogen antibodies and cytokines are single-use consumables andtypically represent the most costly part of the T-cell productionprocess.

Maurice et al. describe the direct engineering of a lentiviral envelopeprotein such that the CD3 agonist OKT3 is displayed on the virionsurface (Maurice et al.; Blood; 2002; 99; 2342-2350). Verhoeyen et al.describe a similar approach in which the lentiviral envelope protein isengineered to incorporate IL7 (Verhoeyen et al.; Blood; 2003; 101;2167-2174).

Each of these engineering approaches requires complex engineering of theviral envelope protein. This complex engineering must be performed foreach discrete peptide to be displayed on the virion surface. Theapproach has also been shown to reduce viral titre.

There is thus a need for new approaches for generating engineered T cellproducts which are not associated with the disadvantages describedabove.

DESCRIPTION OF THE FIGURES

FIG. 1 —Diagram of a retroSTIM vector surrounded by a lipid bilayerwhich is studded with the RD114 envelope glycoprotein and variousmitogenic elements such as scFv or membrane-bound cytokines.

FIG. 2 —Demonstration that an OKT3 scFv can be incorporated into alentivirus. Results show activation of T cells (a)non-stimulated—transduced with lentiviral vector from 293T cells; (b)stimulated with OKT3, CD28.2 and IL2—transduced with lentiviral vectorfrom 293T cells; (c) non-stimulated—transduced with supernatant from293T.OKT3, transfected with only the transfer vector; (d)non-stimulated—transduced with lentiviral vector from 293T.OKT3. Toppanel shows scatter-plots of transduction (x-axis), and activation byCD25 expression (y-axis). Bottom panel show photomicrographs of T-cellcultures. Clumping indicated activation.

FIG. 3 —Demonstration that mitogenic stimulation and transduction of Tcells is dependent on gagpol. 293T cells stably expressing surface boundOKT3 were transfected with gagpol, RD-PRO env, the transfer vector orall three plasmids along with rev. The subsequent supernatant wasapplied to primary human T-cells. The T-cells were studied byflow-cytometry with the following parameters: CD25 to measure T-cellactivation; anti-Fc to detect transgene which was a CAR with an Fcspacer; ki67 to determine cells in cycle. Only conditions where gagpolwas supplied resulted in significant mitogenic stimulation. Only thecondition where all plasmids were supplied (along with rev) resulted inmitogenic stimulation of T-cells and transduction.

FIG. 4 —Demonstration that different lentiviral pseudotyping supportsthe mitogenic effect. 293T cells stably expressing the membrane boundOKT3 were transfected with a lentiviral transfer vector, lentiviralgagpol, rev and different env plasmids: namely VSV-G, RD-PRO, Ampho,GALV and Measles M/H. The subsequent supernatant was applied to primaryhuman T-cells. The cells were subsequently stained with ki67 and studiedby flow-cytometry. All pseudotypes supported the mitogenic effect,although the effect seemed reduced with Measles pseudotyping.

FIG. 5 —Demonstration that mitogenic stimulation and transduction of Tcells is achieved with a gamma-retroviral vector. 293T cells stablyexpressing membrane bound OKT3 were transfected with a gamma-retroviraltransfer vector coding for a CAR, gamma-retroviral gagpol expressionplasmid and an RD114 expression plasmid. Subsequent supernant wasapplied to primary human T-cells. The T-cells were subsequently stainedwith anti-Fc, anti-CD25 and ki67 and studied by flow-cytometry. Althoughno mitogenic stimulus was applied, T-cells were activated, cycling andwere expressing transgene.

FIG. 6 —Demonstration that two different mitogenic stimuli can beincorporated into the viral vector and that an anti-CD3/TCR stimulusalong with an anti-CD28 stimulus has an improved effect compared toanti-CD3/TCR alone.

FIG. 7 —Low resolution microscopy of T-cells stimulated with differentlentiviral vectors generated from 293T cells expressing differentelements on their cell surface.

FIG. 8 —Activation of CD4 and CD8 T cells following transduction withlentiSTIM vectors displaying different combinations of mitogenic andcytokine peptides. Activation is determined by CD25 expression at120-hours post-transduction.

FIG. 9 —Proliferation of CD4 and CD8 T cells following transduction withlentiSTIM vectors displaying different combinations of mitogenic andcytokine peptides. Proliferation is determined by Ki67 expression at120-hours post-transduction.

FIG. 10 —Expansion of T cells following transduction with lentiSTIMvectors displaying different combinations of mitogenic and cytokinepeptides. Expansion is determined by absolute cell counts at 120-hourspost-transduction.

FIG. 11 —Examining the T cell subset phenotype of PBMCs activated witheither lentiSTIM vectors expressing anti-CD3 and anti-CD28 antibodies,or beads coated with anti-CD3 and anti-CD28 antibodies.NM-LV=non-modified lentivirus; STIM-LV=lentiSTIM vector; Tem=effectormemory T cells; Tcm=central memory T cells; Tscm=stem memory T cells;and Tn=naïve T cells.

SUMMARY OF ASPECTS OF THE INVENTION

The present invention is based on the finding that it is possible toincorporate a mitogenic stimulus, and/or a cytokine stimulus, into aretroviral or lentiviral capsid, such that the virus both activates andtransduces T cells. This removes the need to add vector, mitogen andcytokines. The invention involves including a mitogenic transmembraneprotein and/or a cytokine-based transmembrane protein in the producer orpackaging cell, which get(s) incorporated into the retrovirus when itbuds from the producer/packaging cell membrane. The mitogenictransmembrane protein and/or a cytokine-based transmembrane proteinis/are expressed as a separate cell surface molecule on the producercell rather than being part of the viral envelope glycoprotein. Thismeans that the reading frame of the viral envelope is unaffected, whichtherefore preserves functional integrity and viral titre.

Thus in a first aspect the present invention provides a retroviral orlentiviral vector having a viral envelope which comprises:

-   -   (i) a mitogenic T-cell activating transmembrane protein which        comprises a mitogenic domain and a transmembrane domain; and/or    -   (ii) a cytokine-based T-cell activating transmembrane protein        which comprises a cytokine domain and a transmembrane domain    -   wherein the mitogenic or cytokine-based T-cell activating        transmembrane protein is not part of a viral envelope        glycoprotein

The retroviral or lentiviral vector may comprise a separate viralenvelope glycoprotein, encoded by an env gene.

Thus there is provided a retroviral or lentiviral vector having a viralenvelope which comprises:

-   -   (i) a viral envelope glycoprotein: and    -   (ii) a mitogenic T-cell activating transmembrane protein having        the structure:        M-S-TM    -   in which M is a mitogenic domain; S is an optional spacer and TM        is a transmembrane domain; and/or    -   (iii) a cytokine-based T-cell activating transmembrane protein        which comprises a cytokine domain and a transmembrane domain.

The mitogenic T-cell activating transmembrane protein and/orcytokine-based T-cell activating transmembrane protein are not part ofthe viral envelope glycoprotein. They exist as separate proteins in theviral envelope and are encoded by separate genes.

The mitogenic T-cell activating transmembrane protein may have thestructure:M-S-TM

in which M is a mitogenic domain; S is an optional spacer and TM is atransmembrane domain.

The mitogenic T-cell activating transmembrane protein may bind anactivating T-cell surface antigen such as CD3, CD28, CD134 or CD137. Themitogenic T-cell activating transmembrane protein may comprise anagonist for such an activating T-cell surface antigen.

The mitogenic T-cell activating transmembrane protein may comprise thebinding domain from an antibody such as OKT3, 15E8, TGN1412; or acostimulatory molecule such as OX40L or 41BBL.

The viral vector may comprise two or more mitogenic T-cell activatingtransmembrane proteins in the viral envelope. For example, the viralvector may comprise a first mitogenic T-cell activating transmembraneprotein which binds CD3 and a second mitogenic T-cell activatingtransmembrane protein which binds CD28.

The cytokine-based T-cell activating transmembrane protein may, forexample, comprise a cytokine selected from IL2, IL7 and IL15.

In particular there is provided a retroviral or lentiviral vector havinga viral envelope which comprises:

-   -   (ia) a first mitogenic T-cell activating transmembrane protein        which binds CD3; and    -   (ib) a second mitogenic T-cell activating transmembrane protein        which binds CD28.

There is also provided a retroviral or lentiviral vector having a viralenvelope which comprises:

-   -   (ia) a first mitogenic T-cell activating transmembrane protein        which binds CD3;    -   (ib) a second mitogenic T-cell activating transmembrane protein        which binds CD28; and    -   (ii) a cytokine-based T-cell activating transmembrane protein        which comprises IL2.

There is also provided a retroviral or lentiviral vector having a viralenvelope which comprises:

-   -   (ia) a first mitogenic T-cell activating transmembrane protein        which binds CD3;    -   (ib) a second mitogenic T-cell activating transmembrane protein        which binds CD28;    -   (iia) a cytokine-based T-cell activating transmembrane protein        which comprises IL7; and    -   (iib) a cytokine-based T-cell activating transmembrane protein        which comprises IL15.

The viral vector may comprise a heterologous viral envelope glycoproteingiving a pseudotyped viral vector. For example, the viral envelopeglycoprotein may be derived from RD114 or one of its variants, VSV-G,Gibbon-ape leukaemia virus (GALV), or is the Amphotropic envelope,Measles envelope or baboon retroviral envelope glycoprotein.

In a second embodiment of the first aspect of the invention, the viralenvelope of the viral vector may also comprise:

-   -   (iv) a tagging protein which comprises:        -   a binding domain which binds to a capture moiety        -   a spacer; and        -   a transmembrane domain    -   which tagging protein facilitates purification of the viral        vector from cellular supernatant via binding of the tagging        protein to the capture moiety.

The binding domain of the tagging protein may comprise one or morestreptavidin-binding epitope(s). The streptavidin-binding epitope(s) maybe a biotin mimic, such as a biotin mimic which binds streptavidin witha lower affinity than biotin, so that biotin may be used to elutestreptavidin-captured retroviral vectors produced by the packaging cell.

Examples of suitable biotin mimics include: Streptagll (SEQ ID NO: 36),Flankedccstretag (SEQ ID NO: 37) and ccstreptag (SEQ ID NO:38).

The viral vector of the first aspect of the invention may comprise anucleic acid sequence encoding a T-cell receptor or a chimeric antigenreceptor.

The viral vector may be a virus-like particle (VLP).

In a second aspect, the present invention provides a host cell whichexpresses, at the cell surface,

-   -   (ii) a mitogenic T-cell activating transmembrane protein        comprising a mitogenic domain and a transmembrane domain; and/or    -   (ii) a cytokine-based T-cell activating transmembrane protein        which comprises a cytokine domain and a transmembrane domain    -   such that a retroviral or lentiviral vector produced by the        packaging cell is as defined in the first aspect of the        invention.

In a second embodiment of the second aspect of the invention, the hostcell may also express, at the cell surface:

-   -   (iii) a tagging protein which comprises:        -   a binding domain which binds to a capture moiety;    -   and        -   a transmembrane domain    -   which tagging protein facilitates purification of the viral        vector from cellular supernatant via binding of the tagging        protein to the capture moiety,    -   such that a retroviral or lentiviral vector produced by the        packaging cell is as defined in the second embodiment of the        first aspect of the invention.

The tagging protein may also comprise a spacer between the bindingdomain and the transmembrane domain.

The term host cell may be a packaging cell or a producer cell.

A packaging cell may comprise one or more of the following genes: gag,pol, env and/or rev.

A producer cell comprises gag, pol, env and optionally rev genes andalso comprises a retroviral or lentiviral genome.

In this respect, the host cell may be any suitable cell line stablyexpressing mitogenic and/or cytokine transmembrane proteins. It may betransiently transfected with transfer vector, gagpol, env (and rev inthe case of a lentivirus) to produce replication incompetentretroviral/lentiviral vector.

In a third aspect there is provided a method for making a host cellaccording to the second aspect of the invention, which comprises thestep of transducing or transfecting a cell with a nucleic acid encodinga mitogenic T-cell activating transmembrane protein and/or acytokine-based T-cell activating transmembrane protein.

In a fourth aspect there is provided a method for producing a viralvector according to the first aspect of the invention which comprisesthe step of expressing a retroviral or lentiviral genome in a cellaccording to the second aspect of the invention.

In a fifth aspect, there is provided a method for making an activatedtransgenic T-cell or natural killer (NK) cell, which comprises the stepof transducing a T or NK cell with a viral vector according to the firstaspect of the invention, such that the T-cell or NK cell is activated bythe one or more mitogenic T-cell activating transmembrane protein(s)and/or the one or more cytokine-based T-cell activating transmembraneprotein(s).

In a sixth aspect, there is provided a kit for making a retroviral orlentiviral vector as defined in the first aspect of the invention, whichcomprises:

-   -   (i) a host cell as defined in the second aspect of the        invention;    -   (ii) nucleic acids comprising gag, pol, env and optionally rev;        and    -   (iii) a retroviral genome.

There is also provided is provided a kit for making a retroviral orlentiviral vector as defined in the first aspect of the invention, whichcomprises:

-   -   (i) a packaging cell as defined in the second aspect of the        invention; and    -   (ii) a retroviral genome.

There is also provided a kit for making a packaging cell according tothe second embodiment of the second aspect of the invention whichcomprises:

-   -   (i) one or more nucleic acid(s) encoding a mitogenic T-cell        activating transmembrane protein and/or a cytokine-based T-cell        activating transmembrane protein; and    -   (ii) nucleic acids comprising retroviral gag, pol and env genes.

There is also provided a kit for making a producer cell according to thesecond aspect of the invention, which comprises:

-   -   (i) one or more nucleic acid(s) encoding a mitogenic T-cell        activating transmembrane protein and/or a cytokine-based T-cell        activating transmembrane protein;    -   (ii) nucleic acids comprising retroviral gag, pol and env genes;        and    -   (iii) a retroviral or lentiviral vector genome

The invention therefore provides a viral vector with a built-inmitogenic stimulus and/or cytokine stimulus (see FIG. 1 ). The vectorhas the capability to both stimulate the T-cell and to also effect geneinsertion. This has a number of advantages: (1) it simplifies theprocess of T-cell engineering, as only one component needs to be added;(2) it avoids removal of beads and the associated reduction in yield asthe virus is labile and does not have to be removed. (3) it reduces thecost of T-cell engineering as only one component needs to bemanufactured; (4) it allows greater design flexibility: each T-cellengineering process will involve making a gene-transfer vector, the sameproduct can also be made with a mitogenic stimulus to “fit” the product;(5) it allows for a shortened production process: in solubleantigen/bead-based approaches the mitogen and the vector are typicallygiven sequentially separated by one, two or sometimes three days, thiscan be avoided with the retroviral vector of the present invention sincemitogenic stimulation and viral entry are synchronized and simultaneous;(6) it is easier to engineer as there is no need to test a lot ofdifferent fusion proteins for expression and functionality; (7) it ispossible to add more than one signal at the same time; and (8) it ispossible to regulate the expression and/or expression levels of eachsignal/protein separately.

Since the mitogenic stimulus and/or cytokine stimulus are provided on amolecule which is separate from the viral envelope glycoprotein,integrity of the viral envelope glycoprotein is maintained and there isno negative impact on viral titre.

DETAILED DESCRIPTION Retroviruses

Retroviruses are double stranded RNA enveloped viruses mainlycharacterized by the ability to “reverse-transcribe” their genome fromRNA to DNA. Virions measure 100-120 nm in diameter and contain a dimericgenome of identical positive RNA strands complexed with the nucleocapsidproteins. The genome is enclosed in a proteic capsid that also containsenzymatic proteins, namely the reverse transcriptase, the integrase andproteases, required for viral infection. The matrix proteins form alayer outside the capsid core that interacts with the envelope, a lipidbilayer derived from the host cellular membrane, which surrounds theviral core particle. Anchored on this bilayer, are the viral envelopeglycoproteins responsible for recognizing specific receptors on the hostcell and initiating the infection process. Envelope proteins are formedby two subunits, the transmembrane (TM) that anchors the protein intothe lipid membrane and the surface (SU) which binds to the cellularreceptors.

Based on the genome structure, retroviruses are classified into simpleretroviruses, such as MLV and murine leukemia virus; or complexretroviruses, such as HIV and EIAV. Retroviruses encode four genes: gag(group specific antigen), pro (protease), pol (polymerase) and env(envelope). The gag sequence encodes the three main structural proteins:the matrix protein, nucleocapsid proteins, and capsid protein. The prosequence encodes proteases responsible for cleaving Gag and Gag-Polduring particle assembly, budding and maturation. The pol sequenceencodes the enzymes reverse transcriptase and integrase, the formercatalyzing the reverse transcription of the viral genome from RNA to DNAduring the infection process and the latter responsible for integratingthe proviral DNA into the host cell genome. The env sequence encodes forboth SU and TM subunits of the envelope glycoprotein. Additionally,retroviral genome presents non-coding cis-acting sequences such as: twoLTRs (long terminal repeats), which contain elements required to drivegene expression, reverse transcription and integration into the hostcell chromosome; a sequence named packaging signal (ψ) required forspecific packaging of the viral RNA into newly forming virions; and apolypurine tract (PPT) that functions as the site for initiating thepositive strand DNA synthesis during reverse transcription. In additionto gag, pro, pol and env, complex retroviruses, such as lentiviruses,have accessory genes including vif, vpr, vpu, nef, tat and rev thatregulate viral gene expression, assembly of infectious particles andmodulate viral replication in infected cells.

During the process of infection, a retrovirus initially attaches to aspecific cell surface receptor. On entry into the susceptible host cell,the retroviral RNA genome is then copied to DNA by the virally encodedreverse transcriptase which is carried inside the parent virus. This DNAis transported to the host cell nucleus where it subsequently integratesinto the host genome. At this stage, it is typically referred to as theprovirus. The provirus is stable in the host chromosome during celldivision and is transcribed like other cellular proteins. The provirusencodes the proteins and packaging machinery required to make morevirus, which can leave the cell by a process known as “budding”.

When enveloped viruses, such as retrovirus and lentivirus, bud out ofthe host cells, they take part of the host cell lipidic membrane. Inthis way, host-cell derived membrane proteins become part of theretroviral particle. The present invention utilises this process inorder to introduce proteins of interest into the envelope of the viralparticle.

Retroviral Vectors

Retroviruses and lentiviruses may be used as a vector or delivery systemfor the transfer of a nucleotide of interest (NOI), or a plurality ofNOIs, to a target cell. The transfer can occur in vitro, ex vivo or invivo. When used in this fashion, the viruses are typically called viralvectors.

In the viral vectors of the present invention, the NOI may encode a Tcell receptor or a chimeric antigen receptor and/or a suicide gene.

Gamma-retroviral vectors, commonly designated retroviral vectors, werethe first viral vector employed in gene therapy clinical trials in 1990and are still one of the most used. More recently, the interest inlentiviral vectors, derived from complex retroviruses such as the humanimmunodeficiency virus (HIV), has grown due to their ability totransduce non-dividing cells. The most attractive features of retroviraland lentiviral vectors as gene transfer tools include the capacity forlarge genetic payload (up to 9 kb), minimal patient immune response,high transducing efficiency in vivo and in vitro, and the ability topermanently modify the genetic content of the target cell, sustaining along-term expression of the delivered gene.

The retroviral vector can be based on any suitable retrovirus which isable to deliver genetic information to eukaryotic cells. For example,the retroviral vector may be an alpharetroviral vector, agammaretroviral vector, a lentiviral vector or a spumaretroviral vector.Such vectors have been used extensively in gene therapy treatments andother gene delivery applications.

The viral vector of the present invention may be a retroviral vector,such as a gamma-retroviral vector. The viral vector may be based onhuman immunodeficiency virus.

The viral vector of the present invention may be a lentiviral vector.The vector may be based on a non-primate lentivirus such as equineinfectious anemia virus (EIAV).

The viral vector of the invention comprises a mitogenic T-cellactivating transmembrane protein and/or a cytokine-based T-cellactivating transmembrane protein in the viral envelope, as illustratedin FIG. 1 .

The mitogenic T-cell activating transmembrane protein and/orcytokine-based T-cell activating transmembrane protein is/are derivedfrom the host cell membrane, as explained above.

Virus-Like Particles (VLPs)

For retroviral and lentiviral vectors, the expression of the Gagprecursor is sufficient to mediate virion assembly and release. Gagproteins, and even fragments of Gag, have been shown competent toassemble in vitro to form various structures that resemble virion cores.These particles that are devoid of viral genetic material, and are hencenon-infectious, are called virus-like particles (VLPs). Like withcomplete viral particles they contain an outer viral envelope made ofthe host cell lipid-bi-layer (membrane), and hence contain host celltransmembrane proteins.

The viral vector of the first aspect of the invention may be or comprisea virus-like particle.

Nucleotide of Interest (NOI)

The viral vector of the present invention is capable of delivering anucleotide of interest (NOI) to a target cell, such as a T cell or anatural killer (NK) cell.

The NOI may encode all or part of a T-cell receptor (TCR) or a chimericantigen receptor (CAR) and/or a suicide gene.

CARs, are chimeric type I trans-membrane proteins which connect anextracellular antigen-recognizing domain (binder) to an intracellularsignalling domain (endodomain). The binder is typically a single-chainvariable fragment (scFv) derived from a monoclonal antibody (mAb), butit can be based on other formats which comprise an antibody-like antigenbinding site. A spacer domain is usually necessary to isolate the binderfrom the membrane and to allow it a suitable orientation. Atrans-membrane domain anchors the protein in the cell membrane. A CARmay comprise or associate with an intracellular T-cell signalling domainor endodomain.

CAR-encoding nucleic acids may be transferred to cells, such a T cells,using the retroviral or lentiviral vector of the present invention. Inthis way, a large number of cancer-specific T cells can be generated foradoptive cell transfer. When the CAR binds the target-antigen, thisresults in the transmission of an activating signal to the T-cell it isexpressed on. Thus the CAR directs the specificity and cytotoxicity ofthe T cell towards tumour cells expressing the targeted antigen.

A suicide gene encodes a polypeptide which enable the cells expressingsuch a polypeptide to be deleted, for example by triggering apoptosis.An example of a suicide gene is described in WO2013/153391.

Host Cell

In a second aspect, the invention provides a host cell which expresses amitogenic T-cell activating transmembrane protein or a cytokine-basedT-cell activating transmembrane protein at the cell surface.

The host cell may be for the production of viral vectors according tothe first aspect of the invention.

The host cell may be a packaging cell and comprise one or more of thefollowing genes: gag, pol, env and rev.

A packaging cell for a retroviral vector may comprise gag, pol and envgenes.

A packaging cell for a lentiviral vector may comprises gag, pol, env andrev genes.

The host cell may be a producer cell and comprise gag, pol, env andoptionally rev genes and a retroviral or lentiviral vector genome.

In a typical recombinant retroviral or lentiviral vector for use in genetherapy, at least part of one or more of the gag-pol and env proteincoding regions may be removed from the virus and provided by thepackaging cell. This makes the viral vector replication-defective as thevirus is capable of integrating its genome into a host genome but themodified viral genome is unable to propagate itself due to a lack ofstructural proteins.

Packaging cells are used to propagate and isolate quantities of viralvectors i.e to prepare suitable titres of the retroviral vector fortransduction of a target cell.

In some instances, propagation and isolation may entail isolation of theretroviral gagpol and env (and in the case of lentivirus, rev) genes andtheir separate introduction into a host cell to produce a packaging cellline. The packaging cell line produces the proteins required forpackaging retroviral DNA but it cannot bring about encapsidation due tothe lack of a psi region. However, when a recombinant vector carrying apsi region is introduced into the packaging cell line, the helperproteins can package the psi-positive recombinant vector to produce therecombinant virus stock.

A summary of the available packaging lines is presented in“Retroviruses” (1997 Cold Spring Harbour Laboratory Press Eds: J MCoffin, S M Hughes, H E Varmus pp 449).

Packaging cells have also been developed in which the gag, pol and env(and, in the case of lentiviral vectors, rev) viral coding regions arecarried on separate expression plasmids that are independentlytransfected into a packaging cell line, so that three recombinant eventsare required for wild type viral production.

Transient transfection avoids the longer time required to generatestable vector-producing cell lines and is used if the vector orretroviral packaging components are toxic to cells. Components typicallyused to generate retroviral/lentivial vectors include a plasmid encodingthe Gag/Pol proteins, a plasmid encoding the Env protein (and, in thecase of lentiviral vectors, the rev protein), and theretroviral/lentiviral vector genome. Vector production involvestransient transfection of one or more of these components into cellscontaining the other required components.

The packaging cells of the present invention may be any mammalian celltype capable of producing retroviral/lentiviral vector particles. Thepackaging cells may be 293T-cells, or variants of 293T-cells which havebeen adapted to grow in suspension and grow without serum.

The packaging cells may be made by transient transfection with

a) the transfer vector

b) a gagpol expression vector

c) an env expression vector. The env gene may be a heterologous,resulting in a pseudotyped retroviral vector. For example, the env genemay be from RD114 or one of its variants, VSV-G, the Gibbon-apeleukaemia virus (GALV), the Amphotropic envelope or Measles envelope orbaboon retroviral envelope glycoprotein.

In the case of lentiviral vector, transient transfection with a revvector is also performed.

Mitogenic T-Cell Activating Transmembrane Protein

The viral vector of the present invention may comprise a mitogenicT-cell activating transmembrane protein in the viral envelope. Themitogenic T-cell activating transmembrane protein is derived from thehost cell during retroviral vector production. The mitogenic T-cellactivating transmembrane protein is made by the packaging cell andexpressed at the cell surface. When the nascent retroviral vector budsfrom the host cell membrane, the mitogenic T-cell activatingtransmembrane protein is incorporated in the viral envelope as part ofthe packaging cell-derived lipid bilayer.

The term “host-cell derived” indicates that the mitogenic T-cellactivating transmembrane protein is derived from the host cell asdescribed above and is not produced as a fusion or chimera from one ofthe viral genes, such as gag, which encodes the main structuralproteins; or env, which encodes the envelope protein.

Envelope proteins are formed by two subunits, the transmembrane (TM)that anchors the protein into the lipid membrane and the surface (SU)which binds to the cellular receptors. The packaging-cell derivedmitogenic T-cell activating transmembrane protein of the presentinvention does not comprise the surface envelope subunit (SU).

The mitogenic T-cell activating transmembrane protein may comprise oneof the following sequences, or a variant thereof.

(OKT3-CD8STK-TM-A) SEQ ID No. 1METDTLLLWVLLLWVPGSTGQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVV (15E8-CD8STK-TM-A)SEQ ID No. 2 METDTLILWVLLLLVPGSTGQVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWAGGSTNYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCARDKRAPGKLYYGYPDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVSLGQRATISCRASESVEYYVTSLMQVVYQQKPGQPPKLLIYAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQTRKVPSTFGGGTKLEIKRSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRR VCKCPRPVV(TGN1412-CD8STK-TM-A) SEQ ID No. 3METDTLILWVLLLLVPGSTGQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKWYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKRSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVV

The mitogenic T-cell activating transmembrane protein may comprise avariant of the sequence shown as SEQ ID No. 1, 2 or 3 having at least80, 85, 90, 95, 98 or 99% sequence identity, provided that the variantsequence is a mitogenic T-cell activating transmembrane protein havingthe required properties i.e. the capacity to activate a T cell whenpresent in the envelope protein of a retroviral vector.

Methods of sequence alignment are well known in the art and areaccomplished using suitable alignment programs. The % sequence identityrefers to the percentage of amino acid or nucleotide residues that areidentical in the two sequences when they are optimally aligned.Nucleotide and protein sequence homology or identity may be determinedusing standard algorithms such as a BLAST program (Basic Local AlignmentSearch Tool at the National Center for Biotechnology Information) usingdefault parameters, which is publicly available athttp://blast.ncbi.nlm.nih.gov. Other algorithms for determining sequenceidentity or homology include: LALIGN(http://www.ebi.ac.uk/Tools/psa/lalign/ andhttp://www.ebi.ac.uk/Tools/psa/lalign/nucleotide.html), AMAS (Analysisof Multiply Aligned Sequences, athttp://www.compbio.dundee.ac.uk/Software/Amas/amas.html), FASTA(http://www.ebi.ac.uk/TooIs/sss/fasta/) Clustal Omega(http://www.ebi.ac.uk/Tools/msa/clustalo/), SIM(http://web.expasy.org/sim/), and EMBOSS Needle(http://www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html).

The mitogenic T-cell activating transmembrane protein may have thestructure:M-S-TM

in which M is a mitogenic domain; S is an optional spacer domain and TMis a transmembrane domain.

Mitogenic Domain

The mitogenic domain is the part of the mitogenic T-cell activatingtransmembrane protein which causes T-cell activation. It may bind orotherwise interact, directly or indirectly, with a T cell, leading to Tcell activation. In particular, the mitogenic domain may bind a T cellsurface antigen, such as CD3, CD28, CD134 and CD137.

CD3 is a T-cell co-receptor. It is a protein complex composed of fourdistinct chains. In mammals, the complex contains a CD3γ chain, a CD3δchain, and two CD3ε chains. These chains associate with the T-cellreceptor (TCR) and the ζ-chain to generate an activation signal in Tlymphocytes. The TCR, ζ-chain, and CD3 molecules together comprise theTCR complex.

The mitogenic domain may bind to CD3 ε chain.

CD28 is one of the proteins expressed on T cells that provideco-stimulatory signals required for T cell activation and survival. Tcell stimulation through CD28 in addition to the T-cell receptor (TCR)can provide a potent signal for the production of various interleukins(IL-6 in particular).

CD134, also known as OX40, is a member of the TNFR-superfamily ofreceptors which is not constitutively expressed on resting naïve Tcells, unlike CD28. OX40 is a secondary costimulatory molecule,expressed after 24 to 72 hours following activation; its ligand, OX40L,is also not expressed on resting antigen presenting cells, but isfollowing their activation. Expression of OX40 is dependent on fullactivation of the T cell; without CD28, expression of OX40 is delayedand of fourfold lower levels.

CD137, also known as 4-1BB, is a member of the tumor necrosis factor(TNF) receptor family. CD137 can be expressed by activated T cells, butto a larger extent on CD8 than on CD4 T cells. In addition, CD137expression is found on dendritic cells, follicular dendritic cells,natural killer cells, granulocytes and cells of blood vessel walls atsites of inflammation. The best characterized activity of CD137 is itscostimulatory activity for activated T cells. Crosslinking of CD137enhances T cell proliferation, IL-2 secretion survival and cytolyticactivity.

The mitogenic domain may comprise all or part of an antibody or othermolecule which specifically binds a T-cell surface antigen. The antibodymay activate the TCR or CD28. The antibody may bind the TCR, CD3 orCD28. Examples of such antibodies include: OKT3, 15E8 and TGN1412. Othersuitable antibodies include:

Anti-CD28: CD28.2, 10F3

Anti-CD3/TCR: UCHT1, YTH12.5, TR66

The mitogenic domain may comprise the binding domain from OKT3, 15E8,TGN1412, CD28.2, 10F3, UCHT1, YTH12.5 or TR66.

The mitogenic domain may comprise all or part of a co-stimulatorymolecule such as OX40L and 41BBL. For example, the mitogenic domain maycomprise the binding domain from OX40L or 41BBL.

OKT3, also known as Muromonab-CD3 is a monoclonal antibody targeted atthe CD3ε chain. It is clinically used to reduce acute rejection inpatients with organ transplants. It was the first monoclonal antibody tobe approved for clinical use in humans. The CDRs of OKT3 are as follows.

CDRH1: (SEQ ID No. 4) GYTFTRY CDRH2: (SEQ ID No. 5) NPSRGY CDRH3:(SEQ ID No. 6) YYDDHYCLDY CDRL1: (SEQ ID No. 7) SASSSVSYMN CDRL2:(SEQ ID No. 8) DTSKLAS CDRL3: (SEQ ID No. 9) QQWSSNPFT

15E8 is a mouse monoclonal antibody to human CD28. Its CDRs are asfollows:

CDRH1: (SEQ ID No. 10) GFSLTSY CDRH2: (SEQ ID No. 11) WAGGS CDRH3:(SEQ ID No. 12) DKRAPGKLYYGYPDY CDRL1: (SEQ ID No. 13) RASESVEYYVTSLMQCDRL2: (SEQ ID No. 14) AASNVES CDRL3: (SEQ ID No. 15) QQTRKVPST

TGN1412 (also known as CD28-SuperMAB) is a humanised monoclonal antibodythat not only binds to, but is a strong agonist for, the CD28 receptor.Its CDRs are as follows.

CDRH1: (SEQ ID No. 16) GYTFSY CDRH2: (SEQ ID No. 17) YPGNVN CDRH3:(SEQ ID No. 18) SHYGLDWNFDV CDRL1: (SEQ ID No. 19) HASQNIYVLN CDRL2:(SEQ ID No. 20) KASNLHT CDRL3: (SEQ ID No. 21) QQGQTYPYT

OX40L is the ligand for CD134 and is expressed on such cells as DC2s (asubtype of dendritic cells) enabling amplification of Th2 celldifferentiation. OX40L has also been designated CD252 (cluster ofdifferentiation 252).

OX40L sequence (SEQ ID No. 22)MERVQPLEENVGNAARPRFERNKLLLVASVIQGLGLLLCFTYICLHFSALQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL

41BBL is a cytokine that belongs to the tumour necrosis factor (TNF)ligand family. This transmembrane cytokine is a bidirectional signaltransducer that acts as a ligand for 4-1BB, which is a costimulatoryreceptor molecule in T lymphocytes. 41BBL has been shown to reactivateanergic T lymphocytes in addition to promoting T lymphocyteproliferation.

41BBL sequence (SEQ ID No. 23)MEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPS PRSE

Spacer Domain

The mitogenic T-cell activating transmembrane protein and/orcytokine-based T-cell activating transmembrane protein may comprise aspacer sequence to connect the antigen-binding domain with thetransmembrane domain. A flexible spacer allows the antigen-bindingdomain to orient in different directions to facilitate binding.

The spacer sequence may, for example, comprise an IgG1 Fc region, anIgG1 hinge or a human CD8 stalk or the mouse CD8 stalk. The spacer mayalternatively comprise an alternative linker sequence which has similarlength and/or domain spacing properties as an IgG1 Fc region, an IgG1hinge or a CD8 stalk. A human IgG1 spacer may be altered to remove Fcbinding motifs.

Examples of amino acid sequences for these spacers are given below:

SEQ ID No. 24 (hinge-CH2CH3 of human IgG1)AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKD SEQ ID No. 25 (human CD8 stalk):TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDISEQ ID No. 26 (human IgG1 hinge): AEPKSPDKTHTCPPCPKDPKSEQ ID No. 27 (CD2 ectodomain)KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD SEQ ID no. 28 (CD34 ectodomain)SLDNNGTATPELPTQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNEATTNITETTVKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSDLSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGIREVKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLMKKHQSDLKKLGILDFTEQDVA SHQSYSQKT

The spacer sequence may be derived from a human protein. The spacersequence may not be derived from a viral protein. In particular, thespacer sequence may not be, be derived from, or comprise part of thesurface envelope subunit (SU) of a retroviral env protein.

Transmembrane Domain

The transmembrane domain is the sequence of the mitogenic T-cellactivating transmembrane protein and/or cytokine-based T-cell activatingtransmembrane protein that spans the membrane. The transmembrane domainmay comprise a hydrophobic alpha helix. The transmembrane domain may bederived from CD28, which gives good receptor stability.

The transmembrane domain may be derived from a human protein. Thetransmembrane domain may not be derived from a viral protein. Inparticular, the transmembrane domain may not be, be derived from, orcomprise part of the transmembrane envelope subunit (TM) of a retroviralenv protein.

An alternative option to a transmembrane domain is a membrane-targetingdomain such as a GPI anchor.

GPI anchoring is a post-translational modification which occurs in theendoplasmic reticulum. Preassembled GPI anchor precursors aretransferred to proteins bearing a C-terminal GPI signal sequence. Duringprocessing, the GPI anchor replaces the GPI signal sequence and islinked to the target protein via an amide bond. The GPI anchor targetsthe mature protein to the membrane.

The present tagging protein may comprise a GPI signal sequence.

Cytokine-Based T-Cell Activating Transmembrane Protein

The viral vector of the present invention may comprise a cytokine-basedT-cell activating transmembrane protein in the viral envelope. Thecytokine-based T-cell activating transmembrane protein is derived fromthe host cell during viral vector production. The cytokine-based T-cellactivating transmembrane protein is made by the host cell and expressedat the cell surface. When the nascent viral vector buds from the hostcell membrane, the cytokine-based T-cell activating transmembraneprotein is incorporated in the viral envelope as part of the packagingcell-derived lipid bilayer.

The cytokine-based T-cell activating transmembrane protein is notproduced from one of the viral genes, such as gag, which encodes themain structural proteins, or env, which encodes the envelope protein.

The cytokine-based T-cell activating transmembrane protein may comprisea cytokine domain and a transmembrane domain. It may have the structureC-S-TM, where C is the cytokine domain, S is an optional spacer domainand TM is the transmembrane domain. The spacer domain and transmembranedomains are as defined above.

Cytokine Domain

The cytokine domain may comprise part or all of a T-cell activatingcytokine, such as from IL2, IL7 and IL15. The cytokine domain maycomprise part of the cytokine, as long as it retains the capacity tobind its particular receptor and activate T-cells.

IL2 is one of the factors secreted by T cells to regulate the growth anddifferentiation of T cells and certain B cells. IL2 is a lymphokine thatinduces the proliferation of responsive T cells. It is secreted as asingle glycosylated polypeptide, and cleavage of a signal sequence isrequired for its activity. Solution NMR suggests that the structure ofIL2 comprises a bundle of 4 helices (termed A-D), flanked by 2 shorterhelices and several poorly defined loops. Residues in helix A, and inthe loop region between helices A and B, are important for receptorbinding. The sequence of IL2 is shown as SEQ ID No. 29.

SEQ ID No. 29 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIIS TLT

IL7 is a cytokine that serves as a growth factor for early lymphoidcells of both B- and T-cell lineages. The sequence of IL7 is shown asSEQ ID No. 30.

SEQ ID No. 30 MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH

IL15 is a cytokine with structural similarity to IL-2. Like IL-2, IL-15binds to and signals through a complex composed of IL-2/IL-15 receptorbeta chain and the common gamma chain. IL-15 is secreted by mononuclearphagocytes, and some other cells, following infection by virus(es). Thiscytokine induces cell proliferation of natural killer cells; cells ofthe innate immune system whose principal role is to kill virallyinfected cells. The sequence of IL-15 is shown as SEQ ID No. 31.

SEQ ID No. 31 MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQS FVHIVQMFINTS

The cytokine-based T-cell activating transmembrane protein may compriseone of the following sequences, or a variant thereof:

(membrane-IL7) SEQ ID No. 32MAHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGGGSPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLV ITLYCNHRNRRRVCKCPRPVV(membrane-IL15) SEQ ID No. 33MGLVRRGARAGPRMPRGWTALCLLSLLPSGFMAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCK CPRPVV

The cytokine-based T-cell activating transmembrane protein may comprisea variant of the sequence shown as SEQ ID No. 32 or 33 having at least80, 85, 90, 95, 98 or 99% sequence identity, provided that the variantsequence is a cytokine-based T-cell activating transmembrane proteinhaving the required properties i.e. the capacity to activate a T cellwhen present in the envelope protein of a retroviral or lentiviralvector.

Tagging Protein

The viral envelope of the viral vector may also comprise a taggingprotein which comprises a binding domain which binds to a capture moietyand a transmembrane domain.

The tagging protein may comprise:

-   -   a binding domain which binds to a capture moiety    -   a spacer; and    -   a transmembrane domain.

The tagging protein facilitates purification of the viral vector fromcellular supernatant via binding of the tagging protein to the capturemoiety.

‘Binding domain’ refers to an entity, for example an epitope, which iscapable recognising and specifically binding to a target entity, forexample a capture moiety.

The binding domain may comprise one or more epitopes which are capableof specifically binding to a capture moiety. For example the bindingdomains may comprise at least one, two, three, four or five epitopescapable of specifically binding to a capture moiety. Where the bindingdomain comprises more than one epitope, each epitope may be separated bya linker sequence, as described herein.

The binding domain may be releasable from the capture moiety upon theaddition of an entity which has a higher binding affinity for thecapture moiety compared to the binding domain.

Streptavidin-Binding Epitope

The binding domain may comprise one or more streptavidin-bindingepitope(s). For example, the binding domain may comprise at least one,two, three, four or five streptavidin-binding epitopes.

Streptavidin is a 52.8 kDa protein purified from the bacteriumStreptomyces avidinii. Streptavidin homo-tetramers have a very highaffinity for biotin (vitamin B7 or vitamin H), with a dissociationconstant (Kd)˜10⁻¹⁵ M. Streptavidin is well known in the art and is usedextensively in molecular biology and bionanotechnology due to thestreptavidin-biotin complex's resistance to organic solvents,denaturants, proteolytic enzymes, and extremes of temperature and pH.The strong streptavidin-biotin bond can be used to attach variousbiomolecules to one another or on to a solid support. Harsh conditionsare needed to break the streptavidin-biotin interaction, however, whichmay denature a protein of interest being purified.

The binding domain may be, for example, a biotin mimic. A ‘biotin mimic’may refer to an short peptide sequence (for example 6 to 20, 6 to 18, 8to 18 or 8 to 15 amino acids) which specifically binds to streptavidin.

As described above, the affinity of the biotin/streptavidin interactionis very high. It is therefore an advantage of the present invention thatthe binding domain may comprise a biotin mimic which has a loweraffinity for streptavidin compared to biotin itself.

In particular, the biotin mimic may bind streptavidin with a lowerbinding affinity than biotin, so that biotin may be used to elutestreptavidin-captured retroviral vectors. For example, the biotin mimicmay bind streptavidin with a Kd of 1 nM to 100 uM.

The biotin mimic may comprise a sequence as shown in Table 1.

TABLE 1 Biotin mimicking peptides. name Sequence affinity Long nanotagDVEAWLDERVPLVET 3.6 nM (SEQ ID NO: 39) Short nanotag DVEAWLGAR  17 nM(SEQ ID NO: 40) Streptag WRHPQFGG (SEQ ID NO: 41) streptagII WSHPQFEK 72 uM (SEQ ID NO: 36) SBP-tag MDEKTTGWRGGHVVEGLAGELEQ 2.5 nMLRARLEHHPQGQREP (SEQ ID NO: 42) ccstreptag CHPQGPPC 230 nM(SEQ ID NO: 38) flankedccstreptag AECHPQGPPCIEGRK (SEQ ID NO: 37)

The biotin mimic may be selected from the following group: Streptagll,Flankedccstreptag and ccstreptag.

The binding domain may comprise more than one biotin mimic. For examplethe binding domain may comprise at least one, two, three, four or fivebiotin mimics.

Where the binding domain comprises more than one biotin mimic, eachmimic may be the same or a different mimic. For example, the bindingdomain may comprise two Streptagll biotin mimics separated by a linker(for example as shown by SEQ ID NO: 43) or two Flankedccstreptagseparated by a linker (for example as shown by SEQ ID NO: 44).

(StreptagII-d8-x2) SEQ ID NO: 43 WSHPQFEKSGGGGSPAPRPPTPAPTIASWSHPQFEK(Flankedccstreptag-d8-x2) SEQ ID NO: 44ECHPQGPPCIEGRKSSGGGGSPAPRPPTPAPTIASE CHPQGPPCIEGRKS

Glutathione S-Transferase

The binding domain may comprise a glutathione S-transferase (GST)domain.

GSTs comprise a family of eukaryotic and prokaryotic phase II metabolicisozymes which catalyze the conjugation of the reduced form ofglutathione (GSH) to xenobiotic substrates for the purpose ofdetoxification. The GST family consists of three superfamilies: thecytosolic, mitochondrial, and microsomal (also known as MAPEG) proteins(Udomsinpraser et al. Biochem. J. (2005) 388 (Pt 3): 763-71).

The GST protein has a strong binding affinity for GSH and thisinteraction is commonly used in molecular biology to enable theisolation of a GST-tagged protein from a protein mixture.

An amino acid sequence for GST is shown as SEQ ID NO: 45.

(GST) SEQ ID NO: 45 MGTSLLCWMALCLLGADHADAMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVL FQGPLG

Rituximab-Binding Epitope

The present tagging protein may comprise a binding domain whichcomprises a rituximab-binding epitope (R epitope) and/or a Qbend10epitope (Q epitope).

A rituximab-binding epitope refers to an epitope which specificallybinds rituximab. For example, the rituximab-binding epitope may be basedon the CD20 B-cell antigen.

The Rituximab-binding epitope sequence from CD20 is CEPANPSEKNSPSTQYC(SEQ ID No.46)

Perosa et al (2007, J. Immunol 179:7967-7974) describe a series ofcysteine-constrained 7-mer cyclic peptides, which bear the antigenicmotif recognised by the anti-CD20 mAb Rituximab but have differentmotif-surrounding amino acids. Eleven peptides were described in all, asshown in the following table:

Peptide Insert sequence R15-C acPYANPSLc (SEQ ID No. 47) R3-CacPYSNPSLc (SEQ ID No. 48) R7-C acPFANPSTc (SEQ ID No. 49)R8-, R12-, R18-C acNFSNPSLc (SEQ ID No. 50) R14-CacPFSNPSMc (SEQ ID No. 51) R16-C acSWANPSQc (SEQ ID No. 52) R17-CacMFSNPSLc (SEQ ID No. 53) R19-C acPFANPSMc (SEQ ID No. 54) R2-CacWASNPSLc (SEQ ID No. 55) R10-C acEHSNPSLc (SEQ ID No. 56) R13-CacWAANPSMc (SEQ ID No. 57)

Li et al (2006 Cell Immunol 239:136-43) also describe mimetopes ofRituximab, including the sequence:

(SEQ ID No. 58) QDKLTQWPKWLE.

The polypeptide of the present invention comprises a Rituximab-bindingepitope having an amino acid sequence selected from the group consistingof SEQ ID No. 46-58 or a variant thereof which retains Rituximab-bindingactivity.

QBend10

The CliniMACS CD34 selection system utilises the QBEnd10 monoclonalantibody to achieve cellular selection. The present inventors havepreviously mapped the QBEnd10-binding epitope from within the CD34antigen (see WO 2013/153391) and determined it to have the amino acidsequence shown as SEQ ID No. 59.

(SEQ ID No. 59) ELPTQGTFSNVSTNVS.

The binding domain of the present tagging protein the present inventionmay comprise a QBEnd10-binding epitope having the amino acid sequenceshown as SEQ ID No. 59 or a variant thereof which retainsQBEnd10-binding activity.

RQR8

The tagging protein may comprise a binding domain which comprises orconsists of 136 amino acid sequence shown as SEQ ID NO: 60.

(RQR8) SEQ ID NO: 60 CPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVV

Nucleic Acid

The invention also relates to a nucleic acid encoding a cytokine-basedT-cell activating transmembrane protein or a nucleic acid encoding amitogenic T-cell activating transmembrane protein. The nucleic acid maybe in the form of a construct comprising a plurality of sequencesencoding a mitogenic T-cell activating transmembrane protein and/or acytokine-based T-cell activating transmembrane protein.

As used herein, the terms “polynucleotide”, “nucleotide”, and “nucleicacid” are intended to be synonymous with each other.

It will be understood by a skilled person that numerous differentpolynucleotides and nucleic acids can encode the same polypeptide as aresult of the degeneracy of the genetic code. In addition, it is to beunderstood that skilled persons may, using routine techniques, makenucleotide substitutions that do not affect the polypeptide sequenceencoded by the polynucleotides described here to reflect the codon usageof any particular host organism in which the polypeptides are to beexpressed.

Nucleic acids may comprise DNA or RNA. They may be single-stranded ordouble-stranded. They may also be polynucleotides which include withinthem synthetic or modified nucleotides. A number of different types ofmodification to oligonucleotides are known in the art. These includemethylphosphonate and phosphorothioate backbones, addition of acridineor polylysine chains at the 3′ and/or 5′ ends of the molecule. For thepurposes of the use as described herein, it is to be understood that thepolynucleotides may be modified by any method available in the art. Suchmodifications may be carried out in order to enhance the in vivoactivity or life span of polynucleotides of interest.

The terms “variant”, “homologue” or “derivative” in relation to anucleotide sequence include any substitution of, variation of,modification of, replacement of, deletion of or addition of one (ormore) nucleic acid from or to the sequence.

The nucleic acid may produce a polypeptide which comprises one or moresequences encoding a mitogenic T-cell activating transmembrane proteinand/or one or more sequences encoding a cytokine-based T-cell activatingtransmembrane protein. The cleavage site may be self-cleaving, such thatwhen the polypeptide is produced, it is immediately cleaved into thereceptor component and the signalling component without the need for anyexternal cleavage activity.

Various self-cleaving sites are known, including the Foot-and-Mouthdisease virus (FMDV) 2a self-cleaving peptide and various variants and2A-like peptides. The peptide may have the sequence shown as SEQ ID No.34 or 35.

SEQ ID No. 34 RAEGRGSLLTCGDVEENPGP. SEQ ID No 35 QCTNYALLKLAGDVESNPGP

The co-expressing sequence may be an internal ribosome entry sequence(IRES).

The co-expressing sequence may be an internal promoter.

Vector

The present invention also provides a vector, or kit of vectors whichcomprises one or more sequences encoding a mitogenic T-cell activatingtransmembrane protein and/or one or more sequences encoding acytokine-based T-cell activating transmembrane protein. Such a vectormay be used to introduce the nucleic acid sequence(s) into a host cell,such as a producer or packaging cell.

The vector may, for example, be a plasmid or a viral vector, such as aretroviral vector or a lentiviral vector, or a transposon based vectoror synthetic mRNA.

The vector may be capable of transfecting or transducing a host cell.

Method

The invention also provides a method for making an activated transgenicT-cell or natural killer (NK) cell, which comprises the step oftransducing a T or NK cell with a retroviral or lentiviral vectoraccording to the invention, such that the T-cell or NK cell is activatedby one or more mitogenic T-cell activating transmembrane protein(s) andoptionally one or more cytokine-based T-cell activating transmembraneprotein(s).

The method for transducing and activating T cells or NK calls may take48 hours or less, for example between 24 and 48 hours.

The invention will now be further described by way of Examples, whichare meant to serve to assist one of ordinary skill in the art incarrying out the invention and are not intended in any way to limit thescope of the invention.

EXAMPLES Example 1—Production of Viral Vectors Displaying OKT3 on theVirion Surface

An initial proof-of-concept experiment was performed where it wasdemonstrated that expression of an OKT3 scFv on the packaging cellresults the production of viral vector which causes the mitogenicactivation of T-cell targets.

OKT3 scFv 293T cells produced a lentiviral vector which causedactivation and transduction of target T-cells. This mitogenic propertywas dependent on the presence of lentiviral helper components i.e. theeffect was not due to a non-specific property of the packaging cellsupernatant (FIG. 2 ).

A comparison of the OKT3 scFv attached to the membrane via a CD8 stalkor via an IgG1 hinge's ability to incorporate into the lentivirus andcause a mitogenic stimulus was also made, with no difference notedbetween the two spacers.

293T cells stably expressing surface bound OKT3 were transfected withlentiviral gagpol, RD-PRO env, the transfer vector or all three plasmidsalong with a lentiviral rev expressing plasmid. The subsequentsupernatant was applied to primary human T-cells. The T-cells weresubsequently studied by flow-cytometry with the following parameters:CD25 to measure T-cell activation; anti-Fc to detect transgene (CAR withan Fc space)r; ki67 to determine cells in cycle (FIG. 3 ). Onlyconditions where gagpol was supplied resulted in significant mitogenicstimulation.

Only the condition where all plasmids were supplied (along with rev)resulted in mitogenic stimulation of T-cells and transduction

Further experiments were also conducted to determine if differentlentiviral pseudotyping supports the mitogenic effect. 293T cells stablyexpressing the membrane bound OKT3 were transfected with a lentiviraltransfer vector, lentiviral gagpol, rev and different env plasmids:namely VSV-G, RD-PRO, Ampho, GALV and Measles M/H. The subsequentsupernatant was applied to primary human T-cells. The cells weresubsequently stained with ki67 and studied by flow-cytometry. Allpseudotypes supported the mitogenic effect, although the effect seemedreduced with Measles pseudotyping (FIG. 4 ).

Example 2—Two Separate Mitogenic Stimuli can be Incorporated into theViral Vector

An additional construct which comprised the anti-CD28 activating scFvfrom antibody 15E8 was generated. The OKT3 scFv cassette (describedabove) expressed eGFP and the 15E8 scFv cassette expressed the bluefluorescent protein eBFP2.

293T cells were generated which co-expressed high levels of eGFP andeBFP2, demonstrating the successful expression of both OKT3 and 15E8 onthe surface of the 293T cells.

Lentiviral supernatant was generated from wild-type 293T cells, 293Tcells which expressed OKT3 scFv alone and 293T cells which expressedboth OKT3 and 15E8. Activation levels and transduction efficiency wasgreater with the two stimulations (FIG. 6 ).

Example 3—Demonstration of Functionality in Gamma-Retroviral Vectors

293T cells stably expressing membrane bound OKT3 were transfected with agamma-retroviral transfer vector coding for a CAR, gamma-retroviralgagpol expression plasmid and an RD114 expression plasmid. Subsequentsupernant was applied to primary human T-cells. The T-cells were stainedwith anti-Fc, anti-CD25 and ki67 and studied by flow-cytometry. Althoughno mitogenic stimulus was applied, T-cells were activated, cycling andwere expressing transgene (FIG. 5 )

Example 4—Combinations of Peptides Incorporated into Lentivirus Vectors

Different combinations of elements were incorporated into packaging celllines. This included TGN1412 scFv which is a super-agonistic anti-CD28mAb. Cytokines IL7 and IL15, as well as OX40L and 41BBL were alsoincorporated in different combinations as follows:

-   -   1. (Nil)    -   2. OKT3    -   3. OKT3+15E8    -   4. OKT3+ TGN1412    -   5. OKT3+15E8+OX40L+41BBL    -   6. OKT3+15E8+OX40L+41BBL+mIL15    -   7. OKT3+15E8+OX40L+41BBL+mIL7

Lentiviral vector generated from these different 293T cells was used tostimulate/transduce T-cells.

Vector generated from non-engineered 293T cells along with mitogenicsoluble antibodies OKT3 and CD28.2+/−IL2 was used as a control.Activation (CD25), proliferative fraction (Ki67) and absolute counts atday 5 were measured (FIGS. 7-10 ).

It was once again noted that there was a marked advantage ofincorporating two signals instead of one. It was also noted thatactivation using mitogenic peptides displayed on the virion surface wasmarkedly superior to the activation achieved by adding solubleantibodies to the T-cells.

Similar levels of proliferation to that of mAb activation with cytokinewere also achieved.

Methodology

The VH and VL of mitogenic antibodies were cloned as scFvs and connectedto a spacer domain, a TM domain and a polar anchor (SEQ ID Nos 1-3above)

Cytokines were connected in frame to a spacer, a TM domain and a polaranchor (SEQ ID Nos 32 and 33 above).

For native co-stimulatory molecules such as 41BBL and OX40L, these arecloned in their native forms.

Each of the above types of membrane-bound proteins could then be stablyexpressed at high-levels on a 293T cell.

Viral vectors were made from these 293T cells using standard transienttransfection. For lentiviral vector the transfer vector, rev expressionvector, a lenti gagpol expression vector and the RD-PRO expressionvector were co-transfected. For gamma retroviral vectors, the 293T cellswere co-transfected with the transfer vector, MoMLV gagpol and RD114expression plasmid. The supernatant was clarified by centrifugation andfiltration with a 0.45 uM filter. The virus was applied to primary humanT-cells on a retronectin plate. IL2 is added in some conditions, or nocytokines are added in other conditions.

Example 5—Comparing T Cell Subset Phenotypes from Cells Stimulated withLentiviral Vector Versus Cells Stimulated with antiCD3/antiCD28Antibody-Coated Beads

Mononuclear cells were isolated from peripheral blood using standardtechniques. Peripheral blood mononuclear cells (PBMCs) were then treatedwith either:

(i) antiCD3/antiCD28 antibody-coated beads (Dynabeads® Human T-ActivatorCD3/CD28) in a 3:1 ratio in the presence of non-modified lentiviralvector and IL15/IL7; or

(ii) lentiviral vector expressing OKT3 and 15E8B (combination 3 asdescribed in Example 4) on a retronectin-coated plate in the presence ofIL15 and IL7.

After 48 hours, cells were harvested and stained with a panel of T-cellphenotyping antibodies, as follows:

aCD4-BV650

aCD8-PE.Cy7

aC D45 RO-BV605

aCD45RA-FITC

aCD95-PB

aCD197-BV685

T cell subsets were analysed by FACS and the results are summarised inFIG. 11 . For both the CD4+ and CD8+ T cell subsets, the cellsstimulated with virus showed a greater proportion of naïve T cells (Tnand Tscm) than cells stimulated with antibody-coated beads.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology, cellular immunology or related fields are intended tobe within the scope of the following claims.

The invention claimed is:
 1. A retroviral or lentiviral vector having aviral envelope which comprises a cytokine-based T-cell activatingtransmembrane protein which comprises a cytokine domain and atransmembrane domain, wherein the cytokine-based T-cell activatingtransmembrane protein is not part of a viral envelope glycoprotein. 2.The viral vector according to claim 1, which additionally comprises amitogenic T-cell activating transmembrane protein which binds CD3, CD28,CD134 or CD137.
 3. The viral vector according to claim 2, wherein themitogenic T-cell activating transmembrane protein comprises the bindingdomain from OKT3, 15E8, TGN1412, OX40L or 41BBL.
 4. The viral vectoraccording to claim 2, which comprises two or more mitogenic T-cellactivating transmembrane proteins in the viral envelope.
 5. The viralvector according to claim 4, which comprises a first mitogenic T-cellactivating transmembrane protein which binds CD3 and a second mitogenicT-cell activating transmembrane protein which binds CD28.
 6. The viralvector according to claim 1, which comprises a cytokine-based T-cellactivating transmembrane protein which comprises a cytokine selectedfrom IL2, IL7 and IL15.
 7. The viral vector according to claim 1,wherein the viral envelope also comprises: a tagging protein whichcomprises: a binding domain which binds to a capture moiety; and atransmembrane domain, which tagging protein facilitates purification ofthe viral vector from cellular supernatant via binding of the taggingprotein to the capture moiety.
 8. The viral vector according to claim 1which comprises a nucleic acid sequence encoding a T-cell receptor or achimeric antigen receptor.
 9. The viral vector according to claim 1which is a virus-like particle (VLP).
 10. A host cell which expresses,at the cell surface, a cytokine-based T-cell activating transmembraneprotein which comprises a cytokine domain and a transmembrane domain andwhich produces a retroviral or lentiviral vector according to claim 1.11. A host cell according to claim 10, which also expresses, at the cellsurface: a tagging protein which comprises: a binding domain which bindsto a capture moiety and a transmembrane domain, which tagging proteinfacilitates purification of the retroviral or lentiviral vector producedby the host cell from the cellular supernatant via binding of thetagging protein to a capture moiety.
 12. A packaging cell whichexpresses, at the cell surface, a cytokine-based T-cell activatingtransmembrane protein which comprises a cytokine domain and atransmembrane domain and which also comprises one or more of thefollowing genes: gag, pol, env and/or rev.
 13. A producer cell whichexpresses, at the cell surface, a cytokine-based T-cell activatingtransmembrane protein which comprises a cytokine domain and atransmembrane domain and which comprises gag, pol, env and optionallyrev genes and also comprises a retroviral or lentiviral genome.
 14. Amethod for making a host cell according to claim 10 which comprises thestep of transducing or transfecting a cell with a nucleic acid encodinga cytokine-based T-cell activating transmembrane protein.
 15. A methodfor producing a viral vector according to claim 1 which comprises thestep of expressing a retroviral or lentiviral genome in a cell.
 16. Amethod for making an activated transgenic T-cell or natural killer (NK)cell, which comprises the step of transducing a T or NK cell with aviral vector according to claim 1, such that the T-cell or NK cell isactivated by the one or more cytokine-based T-cell activatingtransmembrane protein(s).
 17. A kit for making a retroviral orlentiviral vector according to claim 1, which comprises: (i) a hostcell; (ii) nucleic acids comprising gag, pol, env and optionally rev;and (iii) a retroviral genome.
 18. A kit for making a retroviral orlentiviral vector according to claim 1 which comprises: (i) a packagingcell; and (ii) a retroviral or lentiviral vector genome.
 19. A kit formaking a packaging cell which comprises: (i) one or more nucleic acid(s)encoding a cytokine-based T-cell activating transmembrane protein and(ii) nucleic acids comprising retroviral gag, pol, env and optionallyrev genes.
 20. A kit for making a producer cell which comprises: (i) oneor more nucleic acid(s) encoding a cytokine-based T-cell activatingtransmembrane protein, (ii) nucleic acids comprising retroviral gag,pol, and env and optionally rev genes, and (iii) a retroviral orlentiviral vector genome.